Nitric Oxide
Endogenous nitric oxide (NO) plays a role in the regulation of pulmonary vascular tone. Nelin, et al. Pediatr Res (1994) 35: 20-24; Lipsitz, et al. J Pediatr Surg (1996) 31: 137-140. Nitric oxide is synthesized by different isoforms of the enzyme nitric oxide synthase (NOS). Endothelial NOS (eNOS) is a constitutive enzyme responsible for the calcium-calmodulin dependent production of baseline levels of NO. Inducible NOS (iNOS) catalyzes the calcium-independent production of large amounts of NO in response to certain cytokines and inflammatory stimuli. A third form of NOS is neuronal NOS (nNOS) serves as a neurotransmitter in both the central and peripheral nervous systems. Endothelial cells generate endogenous NO from arginine. Palmer, et al. Biochem Biophys Res Commun (1988) 153: 1251-6; Moncada, et al. N Engl J Med (1993) 329: 2002-12.
The hepatic urea cycle plays a role in the production of two precursors of nitric oxide: arginine and citrulline. Pearson, et al. New England Journal of Medicine (2001) 344: 1832-1838. The first two steps of the hepatic urea cycle, carried out by carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), produce citrulline. These two enzymes are located in mitochondria of the liver and gut with the remainder of the pathway distributed throughout the body, including the pulmonary vascular endothelium. Summar J Inher Metab Dis (1988) 21(S1): 30-39. Citrulline, an amino acid, is the first intermediate of the urea cycle. After citrulline is transported intracellularly via a selective membrane transporter, it is rapidly converted to arginine by the enzymes argininosuccinate synthetase (AS) and argininosuccinate lyase (AL).
After the rate limiting reaction catalyzed by CPSI, three other urea cycle enzymes participate in arginine formation (FIG. 1). In the next urea cycle step, ornithine transcarbamylase (OTC) combines carabamyl phosphate and ornithine to form citrulline. Citrulline is transported from the mitochondria to the cytoplasm. Argininosuccinate synthetase (AS) is the first of the cytoplasmic urea cycle enzymes and combines citrulline with aspartate to form argininosuccinate. See FIG. 1. Argininosuccinate lyase (AL) cleaves fumarate off of argininosuccinate to form arginine. See FIG. 1.
Citrulline and the Urea Cycle
The urea cycle enzymes argininosuccinic acid synthetase (AS) and argininosuccinic acid lyase (AL) participate in the NO regeneration pathway in endothelial tissues (FIG. 1). The substrate supply for this NO pathway comes from the production of citrulline as part of normal urea cycle function.
Arginine is a basic amino acid synthesized predominantly by the urea cycle (FIG. 1). Intracellular concentrations of arginine are many times greater that circulating plasma concentrations, yet NOS function appears to be regulated by plasma concentrations of arginine. Current theory proposes that this phenomenon is due to intracellular co-localization of the arginine transporter, CAT-1, and eNOS in the plasma membrane. CAT-1 uptake of plasma arginine is directly channeled into NO synthesis via eNOS while intracellular arginine stores are separately compartmentalized and unavailable. Both arginine and citrulline can be given orally, however the gut has a partially intact urea cycle and arginase converts much of the dietary arginine to urea. In normal volunteers, oral L-citrulline increases circulating arginine concentrations more than oral arginine.
Human CPSI (carbamoyl phosphate synthetase I) is the rate-limiting enzyme catalyzing the first committed step of ureagenesis in the hepatic urea cycle (FIG. 1). This cycle comprises the body's system for removing waste nitrogen produced by the metabolism of endogenous and exogenous protein. CPSI is highly tissue-specific, with function and production located in the liver and intestine. The product of a nuclear gene, CPSI is synthesized in the cytoplasm and transported into the mitochondria where it is cleaved into its mature 160 kDA monomeric form. The enzyme combines ammonia and bicarbonate to carbamyl phosphate with the expenditure of 2 ATPs and the necessary cofactor n-acetyl-glutamate (NAG). Rubio, et al. Biochemistry (1981) 20: 1969-1974; Rubio, et al. Biochemica Biophysica Acta (1981) 659: 150-160. Mature CPSI is modular in nature, containing 2 main regions. Of particular note is the NAG cofactor-binding domain near the carboxy-terminus of the enzyme. Without NAG binding the enzyme remains inert resulting in hyperammonemia and no citrulline production.
Over 60 CPSI mutations result in disruption of enzyme function. Summar J Inher Metab Dis (1988) 21(S1): 30-39. A common polymorphism is near the 3′ end of the CPSI mRNA (45% heterozygosity). Sequence analysis of this change reveals a C to A transversion at base 4332, changing the triplet code from ACC to AAC. This results in a substitution of asparagine for threonine at amino acid 1405 (referred to as T1405N). The T1405N genotype is associated with pulmonary hypertension, a risk of elevated pulmonary vascular tone in infants and children undergoing correction of their congenital heart defects, and persistent pulmonary hypertension in the newborn (PPHN). Pearson, et al. N Engl J Med (2001) 344(24): 1832-8; Canter, et al. Mitochondrion (2007) 7(3): 204-10.
When arginine and citrulline values were examined in relation to the T1405N alleles, infants with the CC genotype had lower mean arginine and citrulline levels than infants with the AA genotype. Only the arginine values reached statistical significance with a p-value of 0.011. Heterozygous infants (AC genotype) had intermediate levels of arginine and citrulline, which were not statistically different from those of either homozygous group. See FIG. 2
CPSI is the rate-limiting step in the urea cycle, but polymorphisms in other urea cycle enzymes may also affect urea cycle flux and arginine availability. For example, polymorphisms in the enzymes OTC, AS, AL, and eNOS both separately and in combination with CPSI T1405N may have an effect on the postoperative pulmonary vascular tone in infants and children undergoing congenital heart surgery.
The CPSI T1405N genotype affects the postoperative pulmonary outcomes, such as severe pulmonary hypertension, length of mechanical ventilation and length of ICU stay. Thus, supplementing circulatory citrulline levels according to the methods described herein may be expected to improve the postoperative pulmonary outcomes for a patients with CPSI T1405N genotype undergoing cardiac surgery by maintaining the postoperative pulmonary vascular tone.
Pulmonary Vascular Tone
Increased postoperative pulmonary vascular tone (PVT) is an increase in the contraction of smooth muscle in vessel walls. Increased PVT is a common complication after repair of a variety of congenital heart defects. Steinhorn, et al. Artificial Organs (1999) 23: 970-974; Schulze-Neick, et al. J Thorac Cardiovasc Surg (2001) 121: 1033-1039. The pathophysiology of increased postoperative PVT is believed to be involved in pulmonary vascular endothelial cell dysfunction. Steinhorn, et al. Artificial Organs (1999) 23: 970-974. Limited studies have been performed on the effects that cardiopulmonary bypass (CPB) has on pulmonary endothelial function. In a study of 10 infants undergoing CPB for repair of congenital defects, supplementation of the NO precursor, arginine, ameliorated pulmonary endothelial dysfunction. Schulze-Neick, et al. Circulation (1999) 100: 749-755. In animal studies, endothelial cell production of NO is diminished after cardiopulmonary bypass but still a major controlling factor with respect to pulmonary vasomotor tone. Kirshbom, et al. J thorac Cardiovasc Surg (1996) 111: 1248-1256.
Increased pulmonary vascular tone (e.g., excessive contraction) is associated with poor outcomes following specific cardiac surgical procedures for congenital heart defects. Steinhorn, et al. Artificial Organs (1999) 23: 970-974; Russell, et al. Anesthesia and Analgesia 1998; 87:46-51; Yagahi, et al. Artificial Organs 1998; 22:886-891; Zobel, et al. J. of Cardiovascular Surgery 1998; 39:79-86; Bandla, et al. Chest 1999; 116: 740-747; Gamillscheg, et al. J. of Cardiovascular Surgery 1997; 113:435-442; Petrossian, et al. J. of Cardiovascular Surgery 1999; 117:688-695; Amodeo, et al. J. of Cardiovascular Surgery 1997; 114:1020-1031; Gentles, et al. J. of Cardiovascular Surgery 1997; 114:376-391; Freeman, et al. Pediatr Cardiol 1995; 16:297-300; Luciani, et al. Ann Thorac Surg 1996; 61:800-805; Nakajima, et al. Pediatr Cardiol 1996; 17:104-107; Swoonswang, et al. J Am Coll Cardiol 1998; 32:753-757; Weinstein, et al. Circulation 1999; 100S: II-167-11-170; Ishino, et al. J. of Cardiovascular Surgery 1999; 117:920-930; Adatia, et al. J. of Cardiovascular Surgery 1996; 112:1403-1405; Mosca, et al. J. of Cardiovascular Surgery 2000; 119:1110-1118. To some extent, the type of cardiac defect determines the risk for increased pulmonary vascular tone.
AVSD and VSD Repair:
The highest risk patients for postoperative pulmonary hypertension have cardiac defects associated with excess pulmonary blood flow, such as an atrioventricular septal defect (AVSD) or large unrestrictive ventricular septal defect (VSD). Sustained pulmonary overcirculation can cause hypertrophy and hyperreactivity of pulmonary vascular smooth muscle. Preoperatively, these patients often have congestive heart failure and poor weight gain. Surgical repair is scheduled as early as possible for neonates in order to reduce this postoperative complication.
Bidirectional Glenn and Modified Fontan Procedures:
Patients with single ventricle lesions require surgical procedures where success depends on maintenance of low postoperative pulmonary vascular tone. Staged correction of a single ventricle lesion requires a series of three surgical procedures aimed at separating the pulmonary and systemic circulations. The first of these procedures, often performed in the neonatal period, is a Blalock-Taussig shunt for those patients with a hypoplastic right ventricle or a Norwood I procedure for those patients with hypoplastic left heart syndrome. The second surgery is a bidirectional Glenn shunt where superior vena cava (SVC) flow is diverted directly into the pulmonary artery. The third and final stage is a modified Fontan procedure where inferior vena cava (IVC) flow is diverted into the pulmonary artery, thereby completing separation of the pulmonary and systemic circulations. With the Glenn and Fontan procedures, pulmonary blood flow is entirely passive and relies on an adequate pressure gradient between the venous system (SVC and IVC pressure) and the pulmonary artery (PA) pressure. Any elevation in the pulmonary vascular tone in the immediate postoperative period can lead to decreased pulmonary blood flow and a subsequent fall in cardiac output. On a longer term, elevated pulmonary vascular tone after these procedures can lead to persistent pleural effusions, prolonged requirement for pleural or mediastinal drainage tubes, prolonged ventilation, and prolonged ICU stays. Petrossian, et al. J. of Cardiovascular Surgery 1999; 117:688-695; Amodeo, et al. J. of Cardiovascular Surgery 1997; 114:1020-1031; Gentles, et al. J. of Cardiovascular Surgery 1997; 114:376-391.
Arterial Switch Procedure:
Transposition of the great arteries (TGA) is a complex cardiac lesion that requires surgical correction in the immediate neonatal period. Timing of the arterial switch procedure for correction of TGA specifically takes into account pulmonary vascular tone issues. Freeman, et al. Pediatr Cardiol 1995; 16:297-300; Luciani, et al. Ann Thorac Surg 1996; 61:800-805; Nakajima, et al. Pediatr Cardiol 1996; 17:104-107; Swoonswang, et al. J Am Coll Cardiol 1998; 32:753-757. Frequently, surgery is not performed until 5-7 days of age, when perinatal pulmonary vascular tone has partially decreased. Because the right ventricle is the systemic ventricle before surgical correction, postoperative elevations in pulmonary vascular resistance are usually well tolerated and pulmonary artery pressure is usually not measured. However, if postoperative pulmonary vascular tone is increased, it may partially explain why some infants with favorable anatomy and short bypass times still have a complicated postoperative course.
Pulmonary vascular tone can be an important perioperative issue, even for patients not at risk for pulmonary artery hypertension. Protocols for maintaining postoperative pulmonary vascular tone (i.e., reducing or eliminating any increase in PVT) may be helpful in decreasing the need for prolonged mechanical ventilation, ICU stay, and hospitalization. Thus, there exists a need in the art for a more efficient system for maintaining pulmonary vascular tone in a patient during surgery and postoperatively.